CRSQ Archive

Is
Bacterial Resistance to Antibiotics
an Appropriate Example of Evolutionary Change?

Kevin L. Anderson

CRSQ Vol
41 No 4 pp 318-326 March 2005

Abstract

Evolutionists frequently point to the development of antibiotic
resistance by bacteria as a demonstration of evolutionary change. However,
molecular analysis of the genetic events that lead to antibiotic
resistance do not support this common assumption. Many bacteria become
resistant by acquiring genes from plasmids or transposons via horizontal
gene transfer. Horizontal transfer, though, does not account for the
origin of resistance genes, only their spread among bacteria. Mutations,
on the other hand, can potentially account for the origin of antibiotic
resistance within the bacterial world, but involve mutational processes
that are contrary to the predictions of evolution. Instead, such mutations
consistently reduce or eliminate the function of transport proteins or
porins, protein binding afﬁnities, enzyme activities, the proton motive
force, or regulatory control systems. While such mutations can be regarded
as “beneﬁcial,” in that they increase the survival rate of bacteria in the
presence of the antibiotic, they involve mutational processes that do not
provide a genetic mechanism for common “descent with modification.” Also,
some “relative ﬁtness” cost is often associated with such mutations,
although reversion mutations may eventually recover most, if not all, of
this cost for some bacteria. A true biological cost does occur, however,
in the loss of pre-existing cellular systems or functions. Such loss of
cellular activity cannot legitimately be offered as a genetic means of
demonstrating evolution.

Introduction

Because of their rapid rate of replication, ease of
laboratory analysis, and the wide diversity of laboratory-generated
mutants that can be obtained, bacteria have been described as an
excellent model for studying the processes of evolution (Mortlock,
1984). Acquiring resistance to a specific antibiotic provides a clear
benefit to the bacterium when exposed to that antibiotic. Thus, the
acquisition of antibiotic resistance is commonly cited as an example of
"evolutionary change," and has become a popular example of so-called
"evolution in a Petri dish." Miller (1999) refers to the development of
antibiotic resistance as an example of evolution’s "creative force."
Barlow and Hall (2002) refer to it as "the unique opportunity to observe
evolutionary processes over the course of a few decades instead of the
several millennia that are generally required for these processes to
occur." (p. 314)

Evolution is often described simply as ‘change’ or
‘change in gene frequency over time’ (Dillon, 1978; John-son, 2000;
Patterson, 1978), and evolutionists have almost universally maintained
that any change in genotype (or even phenotype) is an "evolutionary
change." As such, any biological change of an organism, including
antibiotic resistance, would fit within this definition. However, mere
biological change also fits within a creation model, and thus this
"vanilla" definition does not readily distinguish itself from creation.
This definition also does not specify the type of change (such as
deleterious versus beneficial), thus it fails to offer any predictive
value to the theory.

What is more, any change that appears to provide a
so-called "beneficial" adaptation is commonly seen as a driving force of
evolution. Indeed, some mutations, such as antibiotic resistance, can be
beneficial since they may provide the organism an increased ability to
survive under very specific environmental conditions. Thus,
evolutionists typically conclude that genetic examples of "evolutionary
change" are abundant and that creationists are forced to deny this
readily observed evidence.

However, the theory of evolution proposes that all
life on Earth had a common origin. Hence, all life shares a common
evolutionary ancestry from which it has descended, i.e., the "common
descent" of life. In a summarizing statement, Darwin (1936) states that
"the theory of descent with modification embraces all the members of the
same great class or kingdom … all animals and plants are descended from
some one prototype." (p. 370). Therefore, through this overall common
"descent with modification," the theory of evolution claims to account
for the origin and diversity of all biological development on Earth.
Thus, common "descent with modification" provides a more appropriate and
functional definition of the theory of evolution, and this article will
refer to evolution in this context. This definition also entails several
"predictions" regarding the types of genetic change necessary for common
evolutionary descent (predictions that are in sharp contrast to the
"predictions" of a creation model). Such changes must provide more than
mere changes in phenotype; they must provide a genetic mechanism that
accounts for the origin of cellular functions and activities (i.e.,
regulatory systems, transport systems, enzyme specificity, protein
binding affinity, etc.).

Genetic changes that reduce or eliminate any of these
cellular systems provide no genetic mechanism for common "descent with
modification." Rather, such changes are actually the antithesis of this
descent, reducing or eliminating a pre-existing system of biological
complexity (a reversal of "descent with modification"). Therefore, these
genetic changes offer no example of a genetic mechanism for the
"evolutionary" acquisition of flight by non-flying organisms, cognition
by non-cognitive organisms, photosynthesis by non-photosynthesizing
organisms, etc. Yet the theory of evolution requires such events to have
occurred, and requires mutations capable of such genetic changes. Hence,
the predictions of evolution require specific types of changes, not just
so-called "beneficial" mutations. Therefore, despite the great claims
that have been made, it is imperative to question whether acquisition of
antibiotic resistance is a valid example of evolutionary change that
supports the predictions of the evolutionary theory (i.e., the theory of
common "descent with modification").

Horizontal Gene Transfer

One means by which bacteria can acquire antibiotic
resistance is via the horizontal transfer of antibiotic resistant genes.
Such transfer of resistance genes is common (Gómez, 1998; Top et al.,
2000), accounting for many examples of resistant bacteria. But,
horizontal transfer merely involves the transfer of resistance genes
already present in the bacterial world.

While horizontal acquisition of resistant genes is
"beneficial" to those bacteria exposed to a given antibiotic, such gene
transfer does not account for the origin or the diverse variety of these
genes. As such, it fails to provide a genetic mechanism for the origin
of any antibiotic resistance genes in the biological world. Evolution,
through the process of common "descent with modification," predicts it
can account for the origin and diversity of life on earth; however, the
mere shuffling of pre-existing genes between organisms via gene transfer
does not provide the necessary genetic mechanism to satisfy this
prediction. Nor can it readily account for the simultaneous development
of both the antibiotic bio-synthesis and resistance genes—an
evolutionary enigma (Penrose, 1998). Thus, horizontal transfer of
resistant genes cannot be offered as an appropriate example of
"evolution in a Petri dish."

Mutations

Mutations, defined as any changes in the DNA sequence
(Snyder and Champness, 2003), provide the only known genetic mechanism
for producing new genetic activity and function in the biological world.
In light of this, only mutations have the potential to provide evolution
a mechanism that accounts for the origin of antibiotic resistance. Thus,
only that resistance resulting from a mutation is a potential example of
"evolution in action" (i.e., common "descent with modification").

In the presence of a particular antibiotic (or other
anti-microbial), any mutation that protects the bacterium from the
lethality of that compound clearly has a "beneficial" phenotype. Natural
selection will strongly and somewhat precisely select for those
resistant mutants, which fits within the framework of an adaptive
response. But, molecular analysis of such mutations reveals a large
inconsistency between the true nature of the mutation and the
requirements for the theory of evolution (Table I).

Bacterial resistance to the antibiotic, rifampin, can
result from a commonly occurring spontaneous mutation. Rifampin inhibits
bacterial transcription by interfering with normal RNA polymerase activity
(Gale et al., 1981; Levin and Hatfull, 1993). Bacteria can acquire resistance
by a point mutation of the â-subunit
of RNA polymerase, which is encoded by the rpoB
gene (Enright et al., 1998; Taniguchi et al., 1996; Wang et al., 2001;
Williams et al., 1998). This mutation sufficiently alters the structure
of the â-subunit so that
it loses specificity for the rifampin molecule. As a result, the RNA
polymerase no longer has an affinity for rifampin, and is no longer
affected by the inhibitory effect of the antibiotic.

In fact, the level of rifampin resistance that a bacterium
can spontaneously acquire can be extremely high. In my laboratory, we
routinely obtain mutant strains with a resistance level that is orders
of magnitude greater than that of the wild-type strain. When rifampin
is present, this mutation provides a decided advantage for survival
com-pared with those cells lacking these specific mutations. But, each
of these mutations eliminates binding affinity of RNA polymerase for
the rifampin. As such, these mutations do not provide a mechanism accounting
for the origin of that binding affinity, only its loss.

Spontaneous resistance to fluoroquinolones (such as
ciprofloxacin or norfloxacin) is also a frequent mutation in some bacteria.
The primary target of the antibiotic is the enzyme, DNA gyrase, which
is comprised of two proteins encoded by the genes, gyrA
and gyrB (Hooper
and Wolfson, 1993). Genetic analysis has found that resistance to this
class of antibiotics can result from a point mutation in either of these
genes (Barnard and Maxwell, 2001; Griggs et al., 1996; Heddle and Maxwell,
2002; Heisig et al., 1993, Willmott and Maxwell, 1993). These mutations
of the gyrase subunits apparently cause a sufficient conformational
change to the gyrase so that its affinity for the fluoroquinolones is
reduced or lost (Figure 1). Again, despite their "beneficial"
nature, these mutations provide no useful model that explains the origin
of the gyrase’s affinity for the fluoroquinolones.

Resistance to streptomycin can also result from spontaneous
bacterial mutations. In this case, streptomycin blocks bacterial protein
synthesis apparently by binding to the 16S rRNA segment of the ribosome
and interfering with ribosome activity (Carter et al., 2000; Leclerc
et al., 1991). Resistance to the antibiotic can occur by mutations in
the 16S rRNA gene, which reduces the affinity of streptomycin for the
16S molecule (Springer et al., 2001). Reduction of specific oligopeptide
transport activities also leads to spontaneous resistance of several
antibiotics, including streptomycin (Kashiwagi et al., 1998). In these
examples, resistance occurred as a result of the loss of a functional
component/activity.

Loss of enzymatic activity can result in metronidazole
resistance. Interacellular metronidazole must be enzymatically activated
before it can serve as an antimicrobial agent. This activation is achieved
by the enzyme, NADPH nitroreductase (Figure 2). If the metronidazole
is not activated it has no inhibitory effect on the bacterium. Thus,
if NADPH nitroreductase activity is absent in the cell metronidazole
remains inactive. Loss of the reductase activity can occur by nonsense
or deletion mutations in rdxA
(Debets-Ossenkopp et al., 1999; Goodwin et al.,
1998; Tankovic et al.,
2000). In addition, NADPH nitroreductase activity can be
severely reduced by a single missense mutation (a single amino acid
change), which reduces its ability to activate metronidazole (Paul et
al., 2001). All these mutations result in loss of the enzyme activity
necessary for the drug to be effective in the cell, hence the cell becomes
resistant to metronidazole. But, loss of enzymatic activity does not
provide a genetic example of how that enzyme originally "evolved."
Hence, mutations that provide resistance to metronidazole cannot be
offered as true examples of "evolution in a Petri dish."

Several bacteria, including Escherichia
coli, construct a multiple-antibiotic-resistance (MAR) efflux
pump that provides the bacterium with resistance to multiple types of
antibiotics, including erythromycin, tetracycline, ampicillin, and nalidixic
acid. This pump expels the antibiotic from the cell’s cytoplasm, helping
to maintain the intracellular levels below a lethal concentration (Grkovic
et al., 2002; Okusu et al., 1996) (Figure 3). The MAR pump is com-posed
of the proteins MarA and MarB, whose synthesis is inhibited by the regulatory
protein, MarR (Alekshun and Levy, 1999; Poole, 2000) (Figure 3). Mutations
that reduce or eliminate the repression control of MarR result in over-production
of the MarAB efflux pump, which enables the cell to expel higher concentrations
of antibiotics or other antibacterial agents (Oethinger et al., 1998;
Poole, 2000; Zarantonelli et al., 1999).

The protein MarA also acts as a positive regulator
by stimulating increased production of both MarA and MarB proteins (Alekshun
and Levy, 1999) [Figure 3]. In addition, the MarA protein indirectly
inhibits the production of the porin, OmpF, a channel in the membrane
that allows entry of some antibiotics into the cell (Cohen et al., 1988).
There-fore, increased expression of MarA increases the efflux of antibiotics
out of the cell, and reduces the transport of some antibiotics into
the cell (Figure 3). Mutations of marR
that reduce expression or activity of the MarR protein will thus enable
over-expression of the MarAB ef.ux pump (Linde et al., 2000; Okusu et
al., 1996), and provide an increased resistance of the bacterium to
various antibiotics (Eaves et al., 2004; Hans-Jorg et al., 2000; Notka
et al., 2002) [Figure 3]. MarR defective mutants also possess increased
bacterial tolerance to some organic chemical agents, such as cyclohexane
(Aono et al., 1998).

Mutations that increase production of this efflux pump
enable these bacteria to survive exposure to various antibiotics. As
such, this is a beneficial mutation when the antibiotic is present in
the environment. However, a mutation that causes loss of regulatory
control (in this case the repressor protein, MarR) does not offer a
genetic mechanism that can account for the origin of this regulatory
control.

In other examples, resistance to erythromycin can also
result from the loss of an 11 base pair segment of the 23S rRNA gene
(Douthwaite et al., 1985), or a mutation that alters the confirmation
of the 23S rRNA—reducing the affinity of the ribosome for the antibiotic
(Gregory and Dahlberg, 1999; Vannuffel et al., 1992). Chloramphenicol
resistance was obtained by deletion of a 12 base pair region in domain
II of the peptidyltransferase gene (Douthwaite, 1992). Resistance to
cephalosporins has been linked to a dramatic alteration of membrane
transport kinetics that is similar to porin-deficient strains (Chevalier
et al., 1999).
Actinonin resistance in Staphylococcus
aureus results from mutations that eliminate expression
of the fmt gene
(Margolis et al., 2000).
Zwittermicin A resistance in
E. coli is associated with loss of proton motive force (Stabb
and Handelsoman, 1998). For
Streptococcus gordonii, penicillin tolerance may involve
loss of regulatory control of the
arc operon (Caldelari et al., 2000). And, E.
coli can survive the presence of ß-lactams, such as ampicillin,
by halting cell division, making the cell less sensitive to the lethal
affect of the antibiotic (Miller et al., 2004).

These resistance mutations described above cause the
loss of a pre-existing biological system, including cell division and
proton motive force. Even though antibiotic survival is a "beneficial"
phenotype, these mutations fail to provide a genetic example of how
each of these systems originated. As such, they fail to provide a genetic
means to fulfill the predictions of "descent with modification."

Resistance to other antibiotics, such as kanamycin,
can result from loss or reduction of synthesis of a transport protein
(OppA) [Kashiwagi et al., 1998]. Ciprofloxacin and imipenem resistance
can result, at least in part, from the decreased formation of the outer
membrane porin, OmpF (Armand-Lefèvre et al.,
2003; Hooper et al., 1987; Yigit et al., 2002). An increase
in meropenem and cefepime resistance is also associated with loss of
OmpF, and another porin, OmpC (Yigit et al.,
2002). And, Enterobacter
aerogenes can become resistant to various antibiotics when
a mutation dramatically reduces the conductance of a membrane porin
(Dé et al., 2001).

Each of these resistances described in the previous
paragraph result from the reduction or loss of a transport system. However,
genetic mechanisms necessary for evolution would need to account for
the origin of these various transport systems. Thus, these antibiotic
resistance mutations do not provide the necessary genetic changes for
"common descent." Rather, they are genetically inconsistent
with the requirements of evolution, each involving the loss of a pre-existing
transport activity.

As a group, the mutations associated with antibiotic
resistance involve the loss or reduction of a pre-existing cellular
function/activity, i.e., the target molecule lost an affinity for the
antibiotic, the antibiotic transport system was reduced or eliminated,
a regulatory system or enzyme activity was reduced or eliminated, etc.
(Table I). These are not mutations that can account for the origin of
those cellular systems and activities. While these mutations would certainly
be "beneficial" for bacterial survival when an antibiotic
is present in the environment, this benefit is at the expense of a previously
existing function. This is analogous to removing an interior wall of
a house to make a larger dining room. While this larger dining room
may be desirable (i.e., beneficial), the mechanism of removing this
wall cannot legitimately be offered as an example of how this interior
wall was originally built. Hence, the survival benefit of a mutation
is only a portion of the genetic characteristics necessary for mutations
to achieve "evolution in a Petri dish." Such mutations must
also provide the genetic basis for common "descent with modification."
While this directly contradicts the claims made by many proponents of
evolution, the molecular data for antibiotic resistance are very clear.

These mutations also cannot provide a mechanism that
continues to "evolve" the level of protein specificity or
protein activity that is necessary for normal cellular function. While
such mutations are excellent examples of bacterial adaptation, they
are actually the antithesis of the mutational change necessary for evolution.
Yet, these are the very examples evolutionists offer as verifiable demonstrations
of "evolutionary change." Ironically, these mutations are,
in fact, verifiable examples of a creation model—initial complexity
being mutated to a level of greater simplicity.

The spontaneous acquisition of antibiotic resistance
is often referred to as "gaining" resistance, but it is more
appropriately identified as a loss of sensitivity. Thus, antibiotic
resistance results from the loss of pre-existing systems in the bacterial
cell. Such changes clearly provide no genetic mechanism for the origin
of such cellular features as enzyme specificity, transport activity,
regulatory activity, or protein binding affinity. Yet, evolutionists
consistently claim that mutations do provide a genetic mechanism for
the origin of biological activity and common "descent with modification,"
and consistently offer the types of mutations described above as examples.

Fitness Cost of Antibiotic Resistance

While mutations that provide resistance to an antibiotic
can be considered "beneficial," they often come with a physiological
cost (Andersson and Levin, 1999; Maisnier-Patin et al., 2002). In fact,
Björkman et al. (2000) conclude that most types of antibiotic resistance
will impart some biological cost to the organism. For example, rifampin
resistance in Mycobacterium
tuberculosis (Billington et al., 1999), E.
coli (Reynolds, 2000), and Staphylococcus
aureus (Wichelhaus et al., 2002) resulted from mutations
to the RNA polymerase that also reduced the relative fitness of most
of the mutant strains. Although the biological cost reported by these
researchers was generally not severe, it was measurable.

This cost of "relative fitness" appears to
vary considerably depending on both the organism and the antibiotic.
Many of the resistant mutants that have been studied, however, including
some of those mentioned above, can subsequently eliminate some or much
of the fitness cost by reversion or suppression mutations, which also
stabilizes the mutation (Andersson and Levin, 1999; Lenski, 1998; Massey
et al., 2001). The degree that a reversion mutation restores fitness
probably depends on the location of the mutation and whether a single
mutation is able to restore some or all of the wild-type "fitness."

Clearly the fitness of some mutant strains is permanently
reduced (sometimes dramatically), and evolutionists have typically ignored
such affects in their rush to promote antibiotic resistance as "evolution
in the Petri dish." In fact, they often test relative fitness of
these mutants under very narrow cultivation parameters, which minimizes
the detectable loss of fitness for a given mutation. On the other hand,
the fitness loss of some mutants is negligible (esp. following reversion
mutations). So, the effect of spontaneous resistance on bacterial fitness
appears to vary from mutant to mutant. Thus, creationists have probably
tended to over-stress the significance of reduced "fitness"
in antibiotic resistant bacteria by applying the concept to all such
mutants.

Resistant mutations do impose a biological cost, though,
in the loss of pre-existing biological systems and activities. Such
biological cost is not compensated by reversion or suppression mutations.
Even though such mutations may not always result in detectable levels
of reduced "fitness," they stand as the antithesis of common
"descent with modification."

Summary

Resistance to antibiotics and other antimicrobials
is often claimed to be a clear demonstration of "evolution in a
Petri dish." However, analysis of the genetic events causing this
resistance reveals that they are not consistent with the genetic events
necessary for evolution (defined as common "descent with modification").
Rather, resistance resulting from horizontal gene transfer merely provides
a mechanism for transferring pre-existing resistance genes. Horizontal
transfer does not provide a mechanism for the origin of those genes.
Spontaneous mutation does provide a potential genetic mechanism for
the origin of these genes, but such an origin has never been demonstrated.
Instead, all known examples of antibiotic resistance via mutation are
inconsistent with the genetic requirements of evolution. These mutations
result in the loss of pre-existing cellular systems/activities, such
as porins and other transport systems, regulatory systems, enzyme activity,
and protein binding. Antibiotic resistance may also impart some de-crease
of "relative fitness" (severe in a few cases), although for
many mutants this is compensated by reversion. The real biological cost,
though, is loss of pre-existing systems and activities. Such losses
are never compensated, unless resistance is lost, and cannot validly
be offered as examples of true evolutionary change.

Douthwaite,
S., J.B. Prince, and H.F. Noller. 1985. Evidence for functional interaction
between domains II and V of 23S ribosomal RNA from an erythromycin-resistant
mutant. Proceedings
of the National Academy of Science 82:8330–8334.

Willmott,
C.J.R., and A. Maxwell. 1993. A single point mutation in the DNA gyrase
A protein greatly reduces the binding of fluoroquinolones to the gyrase-DNA
complex. Antimicrobial
Agents and Chemotherapy
37:126–127.